Establishment of a VVC model in rats

Thirty SD rats were randomly divided into control (n = 6) and experimental groups (n = 24). Rats in the experimental groups were injected vaginally with 0.1 mL of C. albicans suspension (5.0 × 10⁸ yeasts/mL) and the control rats were injected with the same volume of RPMI 1640. The opening to the vaginas was blocked with aseptic cotton balls to prevent the outflow of fluid. At day 4 following inoculation, Gram staining of vaginal swabs from all rats was performed and examined by light microscopy (LM). Vaginal tissues biopsied from some of the infected rats were fixed in 4% paraformaldehyde, paraffin-embedded, sectioned and stained by hematoxylin-eosin (HE) to confirm inflammation. Rats with VVC were identified as those showing symptoms of inflammation and erythema, and having thick white vaginal discharges. Meanwhile, the presence of yeast and hyphae was confirmed by microscopy. Vaginal wash taken from each rat was cultured on SDA containing 50 mg/mL of chloramphenicol at 28 °C for 48 h. The number of yeasts of each animal was counted and expressed as colony-forming units (CFU)/mL at intervals during the vaginal infection.

After VVC confirmation, all rats in the experimental groups were randomly separated into nystatin-treated and untreated groups. To evaluate the time-dependent effects of C. albicans infection, rats in the untreated group were divided into 4 d, 8 d and 15 d sub-groups (n = 6 each group). Rats in treated group were injected vaginally with 2 × 10⁴ units/mL (20 mg/mL) of nystatin suspension for seven consecutive days. The dose of nystatin per day was determined according to animal equivalent dose calculations based on body surface area [31]. Meanwhile, the untreated rats received the same volume of normal saline vehicle (Fig. 1).

Schematic of experiments on C. albicans infection and nystatin treatment in rats. Inf represents Infection

To determine drug efficacy of nystatin, the number of C. albicans (CFU) of vaginal wash was counted and the presence of hyphae or yeast of vaginal secretions was determined by Gram staining under LM in both nystatin-treated group and untreated 15 d sub-group. Samples with no detectable CFU together with no hyphae or yeast as determined by LM were defined as pathogen negative. The negative conversion rate (NCR) was calculated as the number of pathogen negative cases / 6 × 100%.